The role of oxidative BOLENT GORLER, HOSEYIN VURAL,

NEVIN YILMAZ, HALIT OGUZ,

stress in diabetic AHMET SATlel, NURTEN AKSOY

retinopathy

Abstract Key words Diabetes mellitus, Diabetic

retinopathy, Glutathione peroxidase, Lipid
Purpose To investigate the role of oxidative
peroxidation, Superoxide dismutase, Vitamin C
stress in the development of diabetic
retinopathy.
Methods This study included 25 patients with Free radicals (FR), described as atoms and/ or

diabetic retinopathy (group 1),34 patients with molecules with one or more unpaired electrons

non-insulin-dependent diabetes mellitus in the outer orbit, or in the other words reactive

without any angiopathy complications (group oxygen (02) derivatives, continuously form in

II) and 26 healthy subjects (group III). The aerobic organisms as a result of various

serum malondialdehyde (MDA)-like metabolic processes. These highly reactive

metabolite levels as an index of lipid molecules affect biomolecules such as lipids,

12
peroxidation, the erythrocyte glutathione proteins, nucleic acids and carbohydrates, , In

peroxidase (GSH-Px), superoxide dismutase order to compensate the effects of FR, there are

(SOD) and serum vitamin C levels of the defence mechanisms in the organism called

patients and healthy subjects were measured. antioxidants, If the sensitive balance between

Results The mean serum concentration of FR and antioxidant levels is not protected

MDA-like metabolites of patients in group I properly, many pathological alterations occur

was 4.38 ± 1.31 nmollml, in group II was that result in cell damage, and the integrity of
B. Gurler
3.38 ± 0.95 nmollml and in group III was the cell and tissue is threatened. In recent years,
H. Oguz
A. Satici 2.61 ± 0.85 nmollml. There were significant research related to diseases caused by FR has
Department of differences between the groups (p = 0.001 for gained in importance. It is now known that FR
Ophthalmology play an important role in the pathologies such
group I compared with group II, p = 0.0001 for
Harran University
group I compared with group III and p = 0.002 as cancer, atherosclerosis, diabetes mellitus
Faculty of Medicine
Sanliurfa, Turkey for group II compared with group III). There (DM), Parkinson's disease, heart lesions related
was a significant correlation between the to stress, liver pathology, ageing and various
H. Vural types of infectious diseases. These FR are
serum lipid peroxidation concentrations and
N. Aksoy
duration of the disease (r = 0.36,P = 0.047). The continuously formed in all aerobic cells, and
Department of Biochemistry
mean erythrocyte GSH-Px and SOD levels of consist of the superoxide radical (02-'),
Harran University
Faculty of Medicine group I were respectively 68.97 ± 18.04 and hydrogen peroxide (H202) and hydroxyl radical
u
Sanliurfa, Turkey 1597.78 ± 296.46 U/g Hb, of group II were (OH). These metabolites are responsible for
64.30 ±19.26 and 1581.33 ± 278.08 U/g Hb, lipid peroxidation (LPO), which is described as
N. Yilmaz
and of group III were 65.52 ± 17.58 and a conglomeration reaction of the
Department of Internal
Medicine 1587.44 ± 281.17 U/g Hb. There were no polyunsaturated fatty acids found in the cell
Harran University significant differences among the antioxidant membrane (phospholipids, glycolipids,
Faculty of Medicine enzyme levels in the three groups (p > 0.05). glyceride and sterol) to various products such
Sanliurfa, Turkey
The mean serum vitamin C level in group I as peroxides, alcohols, aldehydes, hydroxy fatty

Dr Bulent Gurler � was 42.72 ± 8.90 [Lmolll, in group II was acids, ethane and pentane, In addition, FR
Department of 49.26 ± 11.52 [Lmolll and in group III was damage DNA, inactivate enzymes and oxidise
Ophthalmology 12
58.57 ± 9.75 [Lmoill. There were significant hormones, ,
Harran University
differences among the mean serum vitamin C Antioxidants, described as substances which
Faculty of Medicine
Ara�tirma ve Uygulama
levels of the three groups (p = 0.02 for group I prevent or delay oxidative damage in target

Hastanesi versus group II p = 0.001 for group I versus molecules, may be classified as enzymes or
63200 Sanliurfa, Turkey group III and p = 0.002 for group II versus small non-enzymatic protein molecules
Tel: +90414314 11 70 group III). according to their structure, and as endogenous
Fax: +904143151181 Conclusions Free radicals forming in diabetes or exogenous antioxidants according to their
e-mail: gurlerb@doruk.neUr
mellitus and increasing over time may play a source. In the organism there are many enzymes
role in the development of diabetic with an antioxidant role, such as superoxide
Received: 29 July 1 999
Accepted in revised form: retinopathy, which is an important dismutase (SOD), glutathione peroxidase
13 April 2000 complication of the disease. (GSH-Px), catalase, cytochrome oxidase and

730 Eye (2000) 14, 730-735 © 2000 Royal College of OphthalmologiSts

small molecules such as glutathione, melatonin,
carotenoids, retinoids and vitamins C and E. One of
these, SOD, decreases the intracellular O2- level by
catalysing its transformation to H202 and O2. GSH-Px
reduces H202 and organic hydroperoxides. Vitamin C is
an important component of the cellular defence against
LPO and O2 toxicity caused by FR. It suppresses O2-',
OH and singlet oxygen, and reacts with the tocopheroxyl
radical to re-form tocopherol, and thus prevent LPO.
It has long been known that there is an imbalance
between oxidants and antioxidants in DM. In 1979, Sato
3
et a.1 reported that plasma LPO levels of diabetic
patients are higher than in normal people, and LPO
levels of diabetic patients with angiopathy are higher
than in the other diabetic patients without any
complications. This has been supported by studies
indicating that LPO products increase in the blood of
patients with DM (both type I and type II) and also in
4 21
diabetic rats. - Reactive O2 metabolites, including FR,
increase with auto-oxidation of glucose and glycosylated
proteins in DM, and with the activation of the sorbitol
pathway during hyperglycaemia. Insufficient
neutralisation of FR causes the oxidation of cellular
lipids, proteins, nucleic acids, glycolipids and
glycoproteins? This oxidative effect also causes damage
to the vascular endothelial cells. Additionally,
prostaglandin synthesis is affected due to oxidation of
arachidonic acid, a polyunsaturated fatty acid. Therefore,
it is claimed that the long-term complications of DM are
related to the accumulation of increased FR and LPO
3,4,7 9,12-14,16,17,20
productS. - about 30 min and then centrifuged for 15 min at
This study was planned to research the role of
oxidative stress in diabetic retinopathy (DR). Three
groups of patients were studied: DM cases with DR, DM
cases without DR or any angiopathic complication, and
healthy individuals. The serum malondialdehyde
(MDA)-like metabolites as an index of LPO, erythrocyte
GSH-Px and SOD levels and serum vitamin C
concentration were determined and the data obtained
from these three groups were analysed statistically.
Materials and methods
This study was carried out on 25 patients with DR (group
1),34 patients with non-insulin-dependent DM (NIDDM)
without any angiopathic complication (group II) and 26
healthy subjects (group III). Subjects with DM included
in the study were chosen according to the following
criteria among the subjects with NIDDM who were
under observation in the Internal Medicine Department.
A systemic examination of the subjects, all of them
normotensive, urine analysis for nephropathy,
electrocardiography (ECG) for macrovascular
angiopathic complications and peripheral pulse
examinations were performed to exclude any subjects
from group I who might have any angiopathic
complications other than DR. Those subjects with
proteinuria detected with urine analysis, post­
myocardial infarction and/ or ischaemic symptoms
detected with ECG or whose peripheral pulse was not
taken (confirmed with Doppler ultrasonography) were
considered as having angiopathic complications and
excluded from the study. Subjects with complaints which
may imply diabetic neuropathy such as paresthaesias
and foot pain were also not included in the study. In
fundus examinations of the dilated pupil with direct and
indirect ophthalmoscopy, those with microaneurysms,
dot and/ or blot haemorrhages, or hard and/ or soft
exudates were diagnosed as DR and fundus fluorescein
angiography (FFA) was carried out with a Canon
CF-60UVi. Subjects with DM who had no angiopathic
complication were considered as group II. Group III
consists of the subjects who presented to the Eye Clinic
with no pathology other than a refraction error and had
normal results after systemic examinations. Informed
consent was obtained from all subjects. A history was
taken from all subjects, the time of onset of the disease,
and height and weight measurements. Body mass index
(BMI) was calculated by using the following equation:
2
BMI weight (kg)/length (m ). All subjects were on no
=
other treatment except oral antidiabetics.
Planned biochemical parameters of the subjects who
were chosen according to the criteria mentioned above
were investigated at our Faculty's Department of
Biochemistry Laboratory. Approximately 10 ml of
venous blood was taken from the antecubital vein
following a 12 h fast. Five millilitres of the blood was
transferred to tubes with heparin for the separation of
erythrocytes, and 5 ml to plain tubes for the separation of
serum. Plain tubes were kept at room temperature for
3000 rpm to separate the serum. LPO was investigated
immediately. Heparinised tubes were centrifuged for
10 min at 3000 rpm to separate plasma. An erythrocyte
package was prepared by washing erythrocytes three
times with physiological serum. The erythrocyte package
was stored at -20°C for GSH-Px and SOD analysis.
GSH-Px and SOD activities were measured within a
maximum of 2 weeks.
Serum LPO level was measured using Satoh's method
9,22
based on the reactivity of thiobarbituric acid (TBA).
MDA creates a coloured complex that gives a maximum
absorbance at 532 nm as it reacts with TBA. MDA is a
final product of fatty acid peroxidation and is accepted as
a measure of the LPO index. Serum LPO was given as
nanomoles MDA per millilitre.
GSH-Px activity was measured with a Hitachi 911
automated analyser using a commercial kit (Randox
Lab., UK) in erythrocyte haemolysates. GSH-Px
estimation was based on the following principle: GSH-Px
catalyses the oxidation of glutathione by cumen
hydroperoxide. In the presence of glutathione reductase
and NADPH the oxidised glutathione is immediately
converted to the reduced form with a concomitant
+
oxidation of NADPH to NADP . The decrease in
absorbance at 340 nm is measured. This technique is
15,19,23
based on the method of Paglia and Valentine.
Erythrocyte SOD activity was measured in an
automated analyser (Hitachi 911) using a commercial kit
(Randox Lab., UK). SOD estimation was based on the
731
 Table 1. Sex, a1'erage age, body "ra,;,; i"dex IRMl), fIllA/, a"d dllralio" of di,;C/l,;e of Ihe grollI'';
2
Sex (F1M) Age (years) BMI (kg/m ) HBA!c ( )
"0 Duration of disease (years)
d
Subjects with retinopathy 171R 5·UO :':: 6.R5" 27.5R ':': 4.05 9.14:+: 1.9i' 7.64 :+: 2.R9
(group 1; " = 2S)
Subjects without complications 21 In 51.79 :':: 7.6LJ 26.6LJ :+: 4.39 R.7R :+: 1.46' 3.69 :+: 2.05
(group II; " = 34)

Healthy subjects 1 4 /12 50.23 :+: 6.RLJ 25.71 :+: 3.R2 4.79 � 0.R 2
(group III; " = 26)

Values are the average:+: SO.

"
ap 0.02 in group I compared with group 1lI (Mann-Whitney U-test); 1'
= 0.001 in group I compared with group 1lI; 'p
= 0.001 in group =

1
II compared with group III (Student's I-test); . 1' 0.0001 in group I compared with group II (Mann-Whitney U-test).
=

generation of O2-, produced by xanthine and xanthine and III (p > 0.05), there was a statistically significant
oxidase, which reacts with 2-(4-iodophenyl)-3-(4- difference between groups! and III (p = 0.(2). Although
nitrophenol)-5-phenyltetrazolium chloride to form a red the BM! of group I was higher than that of the other
formazan dye. The SOD activity is then measured by the subjects, there were no statistically significant differences
degree of inhibition of this reaction. This technique is
among the groups (I' > 0.05), Whereas the HbA1c level of
' 2�
used by many investigators.l , .20 Enzyme activities
group! was slightly higher than that of group II, there
were given as units per gram haemoglobin.
were no statistical differences between them (I' > 0.05).
Serum vitamin C level was measured using the
'i,27,2H However, the HbA1c levels of both groups were
dinitrophenylhydrazine method.! Vitamin C in
significantly higher than that of the healthy subjects
serum is oxidised by Cu(lI) to form dehydroascorbic acid
(p 0.001). There was a significant difference (I' 0,0001)
(DHA), which reacts with acidic 2,4-
= =

between the duration of disease in group! (3-16 years)

dinitrophenylhydrazine to form a red bi�-hydrazone,
which is measured at 520 nm. Ascorbic acid (AA) was and group II (6 months to 8 years) All the subjects in the

also used as a standard and the results were expressed as first group, whose fundus examination and FFA were

micromoles per litre. done, had non-proliferative DR Table 2 shows LPO,

Results are given as the average:+: SD, For average
age and duration of disease analysis, the Mann-Whitney
U-test in the SPSS package program was used, and for
other analysis Student's I-test and Spearman correlation
tests were used.
Results
antioxidant enzyme and vitamin C levels of these groups.
LPO assessment of the groups suggested that all
subjects with DM showed an increased LPO, and there
were statistically significant differences between group!
(4.38 :+: 1.31 nmol/ml) and group II (3.38 :+: 0.95 nmol/
ml), group I and group III (2,61 ± 0.85 nmol/ml), and
group II and group III (p 0.0001 and p 0.002,
= =

respectively). There was also a positive relationship

Table 1 shows the sex, age, BM!, HbAlc and duration of
between the duration of disease and LPO in the
disease for 25 subjects with DR of whom 17 were female
correlation analysis (r = 0,36, I' = 0,047). When
and 8 male with ages between 42 and 69 years (average
antioxidant enzyme levels were compared, there were no
54 .60 :+: 6.85 years); 34 subjects with uncomplicated DM
statistically significant differences among the groups
of whom 21 were female and 13 male with ages between
(p > 0.05). Vitamin C levels were low in all subjects with
41 and 66 years (average 51.79 :+: 7.69 years) and 26
DM compared with healthy subjects and there were
healthy subjects of whom 14 were female and 12 male
with ages between 41 and 63 years (average 50.23 ± 6.89 statistically significant differences between group!

years). (42,72 ± 8,90 jJ.mol/l) and group II (49.26 ± 11.52

Regarding the average age of the subjects it was found jJ.mol/I), group! and group III (58.57 ± 9.75 jJ.mol/l) and

that although there was no statistically significant group II and group III (p = 0.02, P = 0.001 and p = 0,002,
difference between groups! and II and between groups II respectively).

Table 2, Saw" lipid peroxidaliml (LPO), erylhrocyfc glllialhiollc I'l'roxida,;c (CSH-i>x), ,;Ill'l'roxide dismula,;c (500) lind serum uilamin C levels
of Ihe gmup,;

LPO (nmol/ml) GSH-Px (U/g Hb) SOD (U/g Hb) Vitamin C (f,lmol/l)

Subjects with retinopathy 4.3R :: 1.31"/' 68,LJ7 ::: 18,()4 ISLJ7.7R ::: 296.46 42.72 ::: R,90d.e
(group I; 11 = 25)

Subjects without complications DR:+: 0,9S' 64.30 :':: 19.26 15RU3 :':: 278. 0 R 49.26 :+: l1.i
(group II; 11 = 34)
Healthy subjects 2,61 :':: O,RS 6S.52 :+: 17.5R 15R7.44 :+: 281.17 SR.57 ::: 9.7S
(group 1II; 11 = 26)

Values are the average:,:: SO.

"I' = 0.001 in group
I compared with group II; /'1' = 0,0001 in group I compared with group 1II; 'p 0. 002 in group II compared with =

0.02 in group I compared with group II; <"I'

group III (Student's I-test); oil' = 0.001 in group I compared with group III; 'I'
= 0.002 in =

group II compared with group III (Student's I-test).

732
Discussion
The balance between the production and destruction of
oxidants formed in cells and tissues has a vital
importance for survival of the structural integrity of
tissues. Inclination of the balance to the side of oxidation
causes cell damage. LPO products causing oxidative
damage are one of the most important endogenous
factors that destroy the lipid layers of cell membranes. In
the studies on the aetiology of DR, it has been
demonstrated that plasma and tissue LPO products in
patients with DM were higher than in healthy persons;
therefore, it has been proposed that increased oxidative
stress is implicated in the aetiology of DM and its long­
4 7 9 12 14 16 17 2 0
term complications?, , - , - , , , In our study it was
found that serum LPO levels of subjects with DR were
higher than in subjects with DM who had no
complications and healthy subjects, the differences
between the levels being statistically quite significant.
Similarly, serum LPO levels of subjects with DM without
any complications were higher than the LPO levels of
healthy subjects, the differences between the levels again
being statistically significant. Our findings were in
21
agreement with those in the literature?- The reasons for
increased LPO in DM are: increased reactive O2 products
as a result of auto-oxidation of glucose and glycosylated
proteins, polyol pathways, and decreased non-enzymatic
antioxidants. In addition, hyperglycaemia increases the
formation of triose phosphate, whose oxidation causes
the formation of two free radicals: alpha oxaldehyde and
17
H202. The increased levels of lipid peroxides can cause
oxidative injury to blood cells, cross-linking of
membrane lipids and proteins, increasing of cell ageing,
imbalance of prostacyclin/prostaglandin and
29
vasoconstriction.
SOD, which belongs to the metallo-proteinase family,
catalyses the dismutation of O2-', into H202. Also
GSH-Px has a key role in enzymatic defence systems and
acts on peroxides (H202 lipid or organic peroxides) to
remove them. In the literature, different results have
been reported in different organs and tissues regarding
the levels of these two enzymes in DM (human and
15 1830 34
experimental). , , - In our study, the erythrocyte SOD
and GSH-Px values were quite similar in each group.
Different results were also reported in the literature
regarding the levels of these two enzymes in the
18,30 35 ,36
erythrocytes of DM subjects. In some studies , the
erythrocyte SOD levels were low, but in others they were
ll 37 38
normal or high. , While in the study by Hagglof et
39
a.l the SOD and GSH-Px activities were low, in the
17
study by Sundaram et al. the SOD activity was low and
GSH-Px activity was high. In accordance with our study,
21 40 41
Ruiz et al., Jos et al. and Walter et al. also found the
erythrocyte SOD and GSH-Px activities similar in DM
and healthy subjects. It is not clear to us whether these
different results are based on different assay methods or
compensatory mechanisms, or on other factors such as
age, duration of diabetes and diabetic state. According to
the authors who found antioxidant enzyme levels low in
DM, because of the inactivation of the antioxidant
enzymes by non-enzymatic glycation due to persistent
36
hyper glycaemia as shown with SOD by Arai et al., the
oxidative stress increase also depends on the
insufficiency of the antioxidant defence system, among
other reasons.
Vitamin C, which is a water-soluble vitamin and the
most important antioxidant vitamin, inactivates FR
found in the cytosol, plasma and extracellular
environment. It is reported that when the FR in plasma
increases, LPO can not occur until all the AA present in
lO
the environment has been oxidised. In healthy tissue
AA always turns to DHA, which returns to AA. If the
regeneration of DHA to AA can not occur, that means if
the AA/DHA ratio decreases, the antioxidant capacity of
the tissue decreases and oxidative damage develops. In
our study, we detected serum AA levels significantly
lower in all DM patients than healthy subjects. In
addition, we found that AA levels of the DR group were
lower than those of the DM group who had no
complications. This result was confirmed by both human
. . 9 17 2842-45 9 43
and ammaI expenments.' , , S'mcI' I ,
a1r et a.
reported that serum AA levels were lower than in
controls, and even lower in DM cases with
microangiopathy. They also emphasised that the ratio of
AA to DHA in DM patients with microangiopathy
decreased compared with both DM patients without
microangiopathy and control groups. Similarly, in the
17
study by Sundaram et a.l it was shown that in DM
when the severity and number of secondary angiopathic
complications increased, the levels of serum vitamin C
gradually decreased. The decreased AA level in DM may
be explained by (a) the enhanced consumption of AA as
a result of increased oxidation of AA to DHA to scavenge
43 45
increased FR , and (b) failed regeneration of AA from
DHA. Intracellular regeneration of AA from DHA may
be impaired because of competitive inhibition of its
transport across the cell membrane by glucose, a
44 46
structurally similar molecule. ,
We have determined a positive relation between LPO
levels and the duration DM. The longer the duration of
DM, the higher the LPO levels (which means more
oxidative stress); this finding shows a parallelism with
the fact that the possibility of DR increases with the
47
duration of DM. This finding was also emphasised by
17
Sundaram et a.l FR in the blood may have a direct
cytotoxic effect on the endothelial cells and cause cellular
1M8
trauma. Moreover it was reported that O2- and OH
increased in the vessel walls of DM and thus the
endothelium-dependent vasodilatation may be impaired
14 49 5D
in diabetes. . , Beside this, inhibition of the
biosynthesis of prostacyclin by lipid peroxides causes
vasoconstriction and proaggregant effects of
thromboxane, so that there may be a tendency to
16
thrombosis.
Endothelial cell destruction has an important role in
microvascular obstruction and leakage, which are basic
pathologies of DR. When it is considered that even a
slight trauma to the endothelial cells of an artery is
48
effective in atherogenesis, in DM, where such trauma
occurs chronically and increases over time as a result of
733
 the effects of LPO products, it is obvious that oxidative
stress plays an important role in the development of
vascular complications.
In conclusion, this study demonstrates significant
abnormalities in oxidant-antioxidant mechanisms in
DM. It was found that the circulating levels of LPO and
vitamin C in patients with DR were higher than in
subjects with DM who had no complications and in
healthy subjects, and that there is a positive correlation
between LPO levels and the duration of DM. In the light
of our findings and additional data in the literature, we
emphasise that FR, which increasingly are produced in
DM, may play a significant role in the development of
DR, one of the important complications of DM.
References
1. Halliwell B. Free radicals, antioxidants, and human disease:
curiosity, cause, or consequence? Lancet 1994;344:721-4.
2. Freeman BA, Crapo JD. Biology of disease, free radicals and
tissue injury. Lab Invest 1982;47:412-26.
3. Sato Y, Hotta N, Sakamoto N, Matsuoka S, Ohishi N, Yagi K.
Lipid peroxide level in plasma of diabetic patients. Biochem
Med 1979;21:104-7.
4. Jennings PE, Jones AF, FIorkowski CM, Lunec I, Barnett AH.
Increased diene conjugates in diabetic subjects with
microangiopathy. Diabet Med 1987;4:452-6.
5. Jain SK, McVie R, Duett I, Herbst JJ. Erythrocyte membrane
lipid peroxidation and glycosylated hemoglobin in diabetes.
Diabetes 1989;38:1539-43.
6. Jain SK, Levine SN, Duett I, Hollier B. Elevated lipid
peroxidation levels in red blood cells of streptozotocin­
treated diabetic rats. Metabolism 1990;39:971-5.
7. Baynes JW. Perspective in diabetes. Role of oxidative stress in
development of complication in diabetes [Review]. Diabetes
1991;40:405-12.
8. Lyons TJ. Oxidised low density lipoproteins: a role in the
pathogenesis of atherosclerosis in diabetes? Diabet Med
1991;8:411-9.
9. Sinclair AJ, Girling AI, Gray L, Lunec J, Barnett AH. An
investigation of the relationship between free radical activity
and vitamin C metabolism in elderly diabetic subjects with
retinopathy. Gerontology 1992;38:268-74.
10. Young IS, Torney JJ, Trimble ER. The effect of ascorbate
supplementation on oxidative stress in the streptozotocin
diabetic rat. Free Rad Bioi Med 1992;13:41-6.
11. Faure P, Corticelli P, Richard MJ, Arnaud I, Coudray C
Halimi S, et al. Lipid peroxidation and trace element status in
diabetic ketotic patients: influence of insulin therapy. Clin
Chern 1993;39:789-93.
12. Augustin AI, Breipohl W, Boker T, Lutz I, Spitznas M.
Increased lipid peroxide levels and myeloperoxidase activity
in the vitreous of patients suffering from proliferative
diabetic retinopathy. Graefes Arch Cli Exp Ophthalmol
1993;231:647-50.
13. Gallau G, Ruelland A, Legras B, Maugendre D, Allannic H,
Cloarec 1. Plasma malondialdehyde in type I and type II
diabetic patients. Clin Chim Acta 1993;214:227-34.
14. Giugliano D, Ceriello A, Paolisso G. Oxidative stress and
diabetic vascular complications. Diabetes Care
1996;19:257-67.
15. Akku� I, Kalak S, Vural H, Caglayan 0, Menek�e E, Can G,
et al. Leukocyte lipid peroxidation, superoxide dismutase,
glutathione peroxidase and serum and leukocyte vitamin C
levels of patients with type II diabetes mellitus. Clin Chim
Acta 1996;244:221-7.
734
16. Losada M, Alio J1. Malondialdehyde serum concentration in
type I diabetic with and without retinopathy. Doc
Ophthalmol 1996;93:223-9.
17. Sundaram RK, Bhaskar A, Vijayalingam S, Viswanathan M,
Mohan R, Shanmugasundaram KR. Antioxidant status and
lipid peroxidation in type II diabetes mellitus with and
without complications. Clin Sci 1996;90:255-60.
18. Yadav P, Sarkar S, Bhatnagar D. Lipid peroxidation and
antioxidant enzymes in erythrocytes and tissues in aged
diabetic rats. Indian J Exp Bioi 1997;35:389-92.
19. Yadav P, Sarkar S, Bhatnagar D. Action of Capparis decidua
against alloxane-induced oxidative stress and diabetes in rat
tissues. Pharmacol Res 1997;36:221-8.
20. Verdejo C, Marco P, Renau-Piqueras I, Pinazo-Duran MD.
Lipid peroxidation in proliferative vitreoretinopathies. Eye
1999;13:183-8.
21. Ruiz C Barbera AR, Farre R, Lagarda MJ. Lipid peroxidation
and antioxidant enzyme activities in patients with type I
diabetes mellitus. Scand J Clin Lab Invest 1999;59:99-106.
22. Satoh K. Serum lipid peroxide in cerebrovascular disorders
determined by a new colorimetric method. Clin Chim Acta
1978;90:37-43.
23. Paglia DE, Valetine WN. Studies on the quantitative and
qualitative characterization of erythrocyte glutathione
peroxidase. J Lab Clin Med 1967;70:158-63.
24. Flohe BL, Otting F. Superoxide dismutase assays. Methods
EnzymoI 1984;105:93-104.
25. Spitz DR, Oberley LW. An assay for superoxide dismutase
activity in mammalian tissue homogenates. Anal Biochem
1989;179:8-18.
26. Guemouri L, Artur Y, Herbeth B. Biological variability of
superoxide dismutase, glutathione peroxidase, and catalase
in blood. Clin Chern 1991;37:1932-7.
27. Omaye ST, Turnbull JD, Sauberlich HE. Selected methods fOI
the determination of ascorbic acid in animal cells, tissues,
and fluids. Methods Enzymol 1979;62:3-11.
28. Stankova L, Riddle M, Larned I, Burry K, Menashe D, Hart J
et al. Plasma ascorbate concentrations and blood cell
dehydroascorbate transport in patients with diabetes
mellitus. Metabolism 1984;33:347-53.
29. Jain SK, McVie R, Jaramillo JI, Palmer M, Smith T, Meachurr
ZD, et al. The effect of modest vitamin E supplementation or
lipid peroxidation products and other cardiovascular risk
factors in diabetic patients. Lipids 1996;31(Suppl):87-90.
30. Loven D, Schedl H, Wilson H, Daabees TT, Stegink LD,
Diekus M, et al. Effect of insulin and oral glutathione on
glutathione levels and superoxide dismutase activities in
organs of rats with streptozotocin-induced diabetes. Diabetes
1986;35:503-7.
31. Godin DV, Wohaieb SA, Garnett ME, Goumeniouk AD.
Antioxidant enzyme alterations in experimental and clinical
diabetes. Mol Cell Biochem 1988;84:223-31.
32. Pereira B, Rosa LFB, Safi DA, Bechara EJH, Curi R.
Superoxide dismutase, catalase and glutathione peroxidase
activities in the lymphoid organs of diabetic rats. J
Endocrinol 1994;142:161-5.
33. Pieper GM, Jordan M, Dondlinger LA, Adams MB, Roza AM.
Peroxidative stress in diabetic blood vessels: reversal by
pancreatic islet transplantation. Diabetes 1995;44:884-9.
34. Kamata K, Kobayashi T. Changes in superoxide dismutase
mRNA expression by streptozotocin-induced diabetes. Br J
Pharmacol 1996;119:583-9.
35. Crouch R, Kimsey G, Priest DG, Sarda A, Buse MG. Effect of
streptozotocin on erythrocyte and retinal superoxide
dismutase. Diabetologia 1978;15:53-7.
36. Arai K, Lizuka S, Tada Y, Oikawa K, Taniguchi N. Increase in
the glucosylated form of erythrocyte Cu-Zn-superoxide
dismutase in diabetes and close association of the
nonenzymatic glucosylation with the enzyme activity.
Biochim Biophys Acta 1987;924:292-6.
37. Kawamura N, Ookawara T, Suzuki K, Konishi K, Mino M,
Taniguchi N. Increased glycated Cu,Zn-superoxide
dismutase levels in erythrocytes of patients with insulin­
dependent diabetes mellitus. J Clin Endocrinol Metab
1992;74:1352-4.
38. Strange RC, Jones P, Bicknell J, Scarpello J. Expression of
Cu,Zn-superoxide dismutase and glutathione peroxidase in
erythrocytes from diabetic and non-diabetic subjects. Clin
Chim Acta 1992;207:261-3.
39. Hagglof B, Marklund SL, Holmgren G. Cu/Zu-superoxide
dismutase, Mn-superoxide dismutase, catalase and
glutathione peroxidase in lymphocytes and erythrocytes in
IDDM. Acta EndocrinoI 1983;102:235-9.
40. Jos J, Rybak M, Patin PH, Robert JJ, Boitard C, Thevenin R.
Antioxidant enzymes in insulin-dependent diabetes in the
child and adolescent. Diabetes Metab 1990;16:498--503.
41. Walter RM, Uriu-Hare JY, Olin KL, Oster MH, Anawalt BD,
Critchfield JW, et al. Copper, zinc, manganese, and
magnesium status and complications of diabetes mellitus.
Diabetes Care 1991;14:1050--6.
42. McLennan S, Yue DK, Fisher E, Capogreco C, Hefferman S,
Ross GR, et al. Deficiency of ascorbic acid in experimental
diabetes: relationship with collagen and polyol pathway
abnormalities. Diabetes 1988;37:359--61.
43. Sinclair AJ, Girling AJ, Gray L, Le Guen C, Lunec J, Barnett
AH. Disturbed handling of ascorbic acid in diabetic patients
with and without microangiopathy during high dose
ascorbate supplementation. Diabetologia 1991;34:171-5.
44. Maxwell SRJ, Thomason H, Leguen DSC, Baxter MA, Thorpe
GHG, Jones AF, et al. Antixodant status in patients with
uncomplicated insulin-dependent and non-insulin­
dependent diabetes mellitus. Eur J Clin Invest
1997;27:484--90.
45. Sun F, Iwaguchi K, Shudo R, Nagaki Y, Tanaka K, Ikeda K,
et al. Change in tissue concentrations of lipid
hydroperoxides, vitamin C and vitamin E in rats with
streptozotocin-induced diabetes. Clin Sci 1999;96:185-90.
46. Bigley R, Wirth M, Layman D, Riddle M, Stankova L.
Interaction between glucose and dehydroascorbate transport
in human neutrophils and fibroblasts. Diabetes
1983;32:545-8.
47. Kanski JJ. Diabetic retinopathy: Retinal vascular disorders.
In: Kanski JJ, editor. Clinical ophthalmology. London:
Butterworth-Heinemann, 1992;300-37.
48. Yagi K. Lipid peroxides and human disease. Chern Phys
Lipids 1987;45:337-51.
49. Pieper GM, Langenstroer P, Siebeneich W. Diabetic-induced
endothelial dysfunction in rat aorta: role of hydroxyl
radicals. Cardiovasc Res 1997;34:145-56.
50. Manas-Rodrigues L, Angulo J, Peiro C, Llergo JL, Sanchez­
Ferrer A, Lopez-Doriga P, et al. Endothelial dysfunction and
metabolic control in streptozotocin-induced diabetic rats. Br J
Pharmacol 1998;123:1495-502.
735